Microtubules and a contractile ring of microfilaments associated with a cleavage furrow

Active contraction occurs in the furrow region of cleaving sea-urchin eggs (Wolpert, 1966). Portions of cortex excised from an incipient furrow region in the ciliate Stentor and grafted elsewhere on the organism’s surface subsequently exhibit contractions and form furrows (Tartar, 1968). Thus it appears that the contractile elements eliciting cleavage are located in the immediate vicinities of furrows. The participation of a contractile ring passing around the base of the cleavage furrow has often been considered (for example, Wolpert, 1960). A constricting ring which apparently pulls the furrow inwards has been described in living grasshopper spermatocytes (Mota, 1959). Rings (or bands, where cleavage is unilateral) of microfilaments, of diameters ranging from 3 to 10 nm, positioned against and running along the furrow base have been described for the ciliate Paramecium (Jurand & Selman, 1969), the cleaving eggs of a diversity of animals (Schroeder, 1968; Tilney & Marsland, 1969; Arnold, 1969; Szollosi, 1970; Selman & Perry, 1970) and dividing epithelial cells of mouse mammary glands (Scott & Daniel, 1970). Densely staining material concentrated along the furrow bases of dividing HeLa (Robbins & Gonatas, 1964) and chick mesenchyme cells (Allenspach & Roth, 1967) probably has the same microfilamentous composition.

The investigation reported here deals mainly with the functional role of a ring of microfilaments situated at the base of the cleavage furrow in the ciliate Nassula during binary fission. Evidence suggesting that this ring actively constricts and that its contraction is the prime cause of the deformations of the organism’s pellicle which occur in the vicinity of the cleavage furrow is presented and discussed. Thousands of micro-tubules are concentrated at the base of the furrow between the pellicle and the ring during the final stages of cleavage; their origin, interaction with the microfilamentous ring, and functional involvement during cleavage are considered.

The species of Nassula used, the procedure for culturing it, and the methods used for electron microscopy and silver staining have already been described (Tucker, 1967, 1970).

The diameters of the cleavage constrictions of several organisms were measured to the nearest 5 μm at intervals after the start of fission (see Tucker, 1970) using a dissecting microscope fitted with an eyepiece micrometer. The organisms remained in the culture dishes at room temperature (22 °C) while these measurements were made, because isolating organisms into depression slides or confining them on microscope slides and beneath coverslips induced abnormal cleavage rates. The sides of furrows are so closely apposed during the later stages of cleavage that furrow bases are not visible using this method. The final measurement in each series was made to the nearest 0·7 μm after the organism had been fixed in an aqueous solution of 25 % glutaraldehyde and 0·6% sucrose buffered at pH 7·8 with phosphate buffer (18 mM). The sides of furrows separated slightly after fixation so that furrow bases could be seen (Fig. 11).

Dividing organisms usually lie motionless on the bottoms of culture dishes. The point at which separation of sister organisms occurred was assessed by directing a current of water at them just sufficient to move the organisms. Separated sisters drifted apart.

The amount of time which had elapsed since the start of fission when organisms were fixed for electron microscopy or silver staining was estimated by comparing the diameters of their cleavage constrictions with those of living organisms measured at known times after the start of fission.

The cleavage furrow constricts the organism at a progressively more rapid rate for about 90 min following the start of fission until the cleavage constriction has a diameter of about 5 /tm (Fig. 1). Then furrow advance slows abruptly, and for a period of about 25 min the diameter of the constriction either remains unchanged or slowly decreases (Fig. 1). These 2 phases of furrowing will be referred to as the rapid and slow phases of cleavage, respectively. Finally the cleavage constriction suddenly stretches, narrows, and breaks (Figs. 4–6).

During the rapid phase of cleavage, and at the start of the slow phase, a ring of microfilaments (f) is positioned underneath the dense pellicular layer (d) at the base of the furrow (Figs. 2, 9). The microfilaments are fairly straight and their longitudinal axes run around the ring. They have diameters ranging from 4 to 10 nm. The diameter of an individual microfilament sometimes varies slightly at different points along its length. Fine bridges (arrows) connect adjacent microfilaments; other dense material of irregular shape and arrangement is also situated between filaments (Fig. 9). Cross-sections of portions of the ring have a reticular appearance; irregularly spaced densely staining nodes are interconnected by strands of less densely staining material (Fig. 7). The nodes are probably microfilaments.

About 100 ciliary rows (kineties) extend between anterior and posterior poles of Nassula. Kineties are drawn closer together in the cleavage constriction as furrowing proceeds (Fig. 8). A bundle of subpellicular microtubules extends along the length of each kinety at the start of fission; the tubules have diameters of about 24 nm. Each bundle consists of about 30 tubules arranged in a series of closely stacked straight rows situated just beneath the dense pellicular layer (d) (Fig. 10). There are no subpellicular tubules in the regions between kineties at the start of fission. Stacks of tubule rows are no longer present in the furrow region by about 85 min after the start of fission. The dense pellicular layer (d) is slightly folded and a curved row of tubules (m) is positioned below each of the larger folds. Densely staining material (i) is associated with the tubules; it forms a more or less continuous layer between the dense pellicular layer and the microfilamentous ring (f) and extends between tubule rows (Fig. 9). The tubules, diameter about 24 nm, have slightly curved longitudinal profiles; they curl around the base of the furrow. Less densely staining regions are included in the layer of densely staining material where it extends between tubule rows. These regions (x) are circular in cross-section and have the same diameters as the cores of the tubules (m) (Fig. 9). They may be developing tubules, their walls at this point having the same density as the surrounding dense material, because at this time, as will be shown below, large numbers of microtubules are forming in the regions occupied by the dense material.

At the end of the rapid phase of cleavage the dense pellicular layer is much more extensively folded than it was previously (compare Figs. 2, 9). Large numbers of sub-pellicular microtubules (m) and associated dense material (i) are concentrated inside the folds forming a girdle between the dense pellicular layer (d) and the microfilamentous ring (f) (Fig. 2). Folds (d) occur only where the dense pellicular layer overlies the ring; their longitudinal axes are at right angles to the longitudinal axes of the micro-filaments (f) in the ring (Fig. 13). The vast majority of the girdle tubules have the same lengths as the folds (about 1·8 μm) and do not protrude beyond the ends of the folds. The cleavage constrictions of 2 organisms fixed during the slow phase of cleavage possessed about 14000 and 12000 girdle tubules (Figs. 2 and 3, respectively). Nassula has about 100 kineties and sections show about 30 tubules in each kinety tubule bundle. Hence bunching of kinety tubules in the cleavage constriction can account at most for only about 3000 girdle tubules. Whether or not any of the kinety tubules contribute to the girdle has not been ascertained. However, even if all the kinety tubules are included in the girdle, development of several thousand new tubules must occur during girdle formation. No spindle tubules are included in the girdle. Spindles (s) still span the cleavage constriction 86 min after the start of fission but they are inside the ring (/) (Fig. 12) and divide before the slow phase of cleavage begins (see Tucker, 1967), so that none of them traverses the constriction during the slow phase of cleavage (Fig. 2).

Portions of the ring (f) have saddle-shaped cross-sectional profiles during the rapid phase of cleavage (Fig. 12). The ring thickens throughout the later stages of the rapid phase of cleavage; it has not been examined during earlier cleavage stages. For example, the ring has a maximum thickness of about 0·15 μm 85 min after the start of fission (Fig. 9) and about 10 min later a maximum thickness of about 0·4 μm (Fig. 2). Whether or not the breadths of the saddle-shaped profiles of the ring also change has not been ascertained. At the beginning of the slow phase of cleavage a considerable proportion of the microfilaments lining the inner surface of the ring (arrows) are no longer oriented with their longitudinal axes passing around the ring (f) (Fig. 2). These regions of the ring have a reticular appearance in cross-sections of the constriction, indicating that the filaments have been cut in transverse or oblique section. Most of the microfilaments (f) have lost their annular arrangement during the later stages of the slow phase of cleavage, although a few (arrows) still pass around the inner surface of the girdle; the microfilaments form a plug completely filling the cleavage constriction interior to the girdle (Fig. 3).

The orientation and positioning of pellicular folds with respect to the ring are precisely those which would be expected if the ring were actively contracting and attached to the overlying pellicle. The progressively more rapid rate at which constriction occurs during the rapid phase of cleavage also indicates that the micro-filamentous ring is the main contractile agent involved in furrow development. For the ring thickens as it constricts and there is no increase in the lateral spacing of micro-filaments as this occurs. If the breadth of the ring does not decrease, then as the ring constricts the number of microfilaments per unit segment of the ring increases. If the tension generated in the ring is proportional to the number of microfilaments per unit ring segment then ring tension and the rate of furrowing should increase progressively as observed (Fig. 1), provided that there is no marked change in the resistance of the pellicle to its deformation, or change in the total number of ring filaments after the start of fission. These observations are also compatible with the view that sliding interactions between overlapping filaments are an essential feature of the tension-generating process and that the rate of furrowing accelerates because more filaments overlap one another per unit ring segment as the ring constricts and thickens.

Microfilaments are associated with cleavage furrows in eggs and tissue cells (see Introduction) where furrowing probably involves mechanisms similar to those examined here in a ciliate. If furrow microfilaments operate in the same way in all animal cells, furrowing may frequently proceed at a progressively more rapid rate. Both Wolpert (1966) and Dan (1963) have recorded an increase in the rate of furrowing during the early stages of cleavage in sea urchin eggs. However, Szollosi (1970) has noted that the thickness of the microfilamentous band at the base of furrows in eggs of the cnidarian Aequorea does not change appreciably during cleavage.

Microfilamentous rings are associated with sphincter-like contractions in other situations. For example, in Nassula a microfilamentous annulus encircles the top of the cytopharynx which constricts when the organism feeds (Tucker, 1968) and bands of microfilaments encircle the accessory hearts of the ascidian Botryllus (de Santo & Dudley, 1969). There is an abundance of reports for a wide range of protozoa, metazoan tissue cell types, and even for certain algae (Drum & Hopkins, 1966; Nagai & Rebhun, 1966; Halfen & Castenholz, 1970) dealing with microfilaments situated in cytoplasmic regions where some form of contraction or movement is taking place. The description of microfilaments, diameter about 7 nm, exhibiting ATPase activity in the slime mould Physarum (Wohlfarth-Bottermann, 1964) is especially important because materials which markedly resemble actin and myosin have been isolated from this organism (Hatano & Tazawa, 1968; Nachmias, Huxley & Kessler, 1970). There are indications both for (Rappaport, 1967) and against (Wolpert, 1963) the possibility that an actomyosin-like complex is involved in furrow formation.

Mitchison (1952) noted that if a contracting ring was involved in furrowing then it would either have to constrict to nothing, which would involve something more than a straightforward contraction of the ring, or the final nipping off of sister cells when the cleavage constriction attained a diameter of less than 5 μm might be due to some mechanism different from that involved in ring constriction. It is clear that in Nassula the ring does not constrict to nothing, but forms a plug in the constriction, and there are 2 distinct phases in the cleavage process, rapid and slow, distinguished by an abrupt decrease in the rate of furrow advance, the onset of pellicular folding and considerable changes in the arrangement and composition of fibrous elements associated with the furrow base when the cleavage constriction has a diameter of about 5 μm.

An abrupt slowing of furrowing and the consequent existence of a relatively persistent cytoplasmic bridge during the final cleavage stages has been reported for a range of animal cell types (Hughes & Swann, 1948; Chambers, 1951; Lewis, 1951). In dividing HeLa cells cleavage proceeds for about 26 min to form a cytoplasmic bridge with a diameter of about 1·5 μm, which then persists for 2 h or longer before a complex sequence of events, quite different from anything observed during earlier cleavage stages, finally severs the bridge (Byers & Abramson, 1968). Very probably, special events quite distinct from, or at least additional to, those concerned with the majority of a furrow’s advance are introduced during the final cleavage stages in most animal cells to expedite the final separation of sister cells and ensure that a fairly durable and impermeable barrier at all times partitions liquid cytoplasm from the extracellular medium in the region of final severance. In Nassula the microfilamentous plug and the microtubular girdle may be involved in sealing the broken ends of the cytoplasmic strand when severance occurs.

No indications of the activity responsible for the final constriction, elongation, and severance of the cleavage constriction during the slow phase of cleavage have been obtained. Active sliding of girdle tubules in the manner suggested for extension of the microtubular km fibres of Stentor (Bannister & Tachell, 1968) and/or attempts by sisters to swim apart may be involved.

Microtubules are generally believed to be fairly rigid structures. This is certainly the case for certain microtubular components in the cytopharynx of Nassula (Tucker, 1968). Hence the formation of the microtubular girdle in the cleavage constriction probably impedes furrow advance to some extent. The girdle starts to develop at the end of the rapid phase of cleavage but considerable constriction and pellicular folding occur after the tubules have formed. Since the girdle lies between the ring and the pellicle any contractions causing furrowing and originating in the ring must be trans-mitted through the girdle to the pellicle. The densely staining material which lies between girdle tubules and extends between the ring and the pellicle may effect such transmission. While binding tubules, ring and pellicle together a substantial amount of shearing and/or flowing occurs in the dense material permitting the ring to constrict and the pellicle to fold. In this respect the dense material behaves as a sticky gel. Densely staining material of similar appearance occupies intertubular spaces and apparently plays a similar role in regions of the cytopharynx of Nassula where the greatest shearing stresses are believed to occur; here too considerable deformations of the dense material sometimes take place (Tucker, 1968).

Accumulation of girdle tubules is not simply a consequence of their being trapped in the cleavage constriction by the advancing furrow, as is apparently the case during formation of the microtubular spindle mid-body in the late cleavage constriction of some tissue cells (for example, Allenspach & Roth, 1967). The development of girdle tubules is precisely correlated in both space and time with final stages in the formation of the cleavage constriction. The girdle therefore probably has an important role concerned with events bringing about the final narrowing and severance of the constriction. Special cleavage tubules are formed in the flagellate Chlamydomonas and it has been suggested that they may provide a rigid framework influencing the form of the furrow (Johnson & Porter, 1968). Girdle tubules in Nassula may act similarly. It has been argued that the greatest contractile forces develop in the ring at the end of the rapid phase of cleavage; this is precisely the time at which the girdle develops. The role of the girdle may be to distribute ring tension in a longitudinal direction relative to the organism’s polar axis, so that a cleavage constriction of some length is formed and the base of the furrow has a U-shaped rather than a V-shaped profile. The girdle may ensure that the cleavage constriction has considerable resistance to breaking before procedures such as the formation of the microfilamentous plug seal the more liquid cytoplasmic portions of the sister cells from each other. The combined actions of the microtubular girdle and the contractile microfilamentous ring may be like the application of a corset in which a system of constricting cords draws in a circular palisade of stays to shape and support a slender waist.

Part of this investigation was undertaken during the tenure of an S.R.C. Research Fellowship which is gratefully acknowledged. I should like to thank Dr M. A. Sleigh for critically reading the manuscript.

Unless otherwise stated, all figures are electron micrographs of Nassula fixed with glutaraldehyde, post-fixed with osmium tetroxide and stained with uranyl acetate and lead citrate. The estimated period of time elapsing after the start of fission when an organism was fixed, or in the case of a living organism when it was photographed, is indicated in the accompanying legend by the period in question followed by the abbreviation ‘a.s.f.’.